Modified cDNA factor VIII and its derivatives

ABSTRACT

Modified human factor VIII cDNA wherein mutations are inserted either in the wild-type factor VIII cDNA or in a factor VIII cDNA in which the B-domain is partially or completely deleted and may be replaced by a DNA linker segment and  
     A) one or several codons of the human factor VIII cDNA which are not identical with the corresponding codon in the same position of the porcine factor VIII cDNA are substituted by a different codon in such a way that  
     when the human sequence contains a codon for a neutral amino acid whereas the porcine sequence contains a codon for a charged amino acid then a codon for an amino acid with the same charge as found in the porcine sequence is introduced into the human sequence;  
     when the human sequence contains a codon for a charged amino acid whereas the porcine sequence contains a codon for a neutral amino acid then a codon for a neutral amino acid or a codon for an amino acid of the opposite charge is introduced into the human sequence,  
     when the human sequence contains a codon for a charged amino acid whereas the porcine sequence contains a codon for an amino acid with the opposite charge then a codon for an amino acid with the opposite charge is introduced into the human sequence or  
     B) one or several codons for a charged amino acid which are found in the FVIII cDNA of a hemophilic patient are replaced by a codon for an amino acid of the opposite charge.

[0001] The present invention relates to modified DNA sequences codingfor biologically active recombinant human factor VIII and itsderivatives with improved stability, recombinant expression vectorscontaining such DNA sequences, host cells transformed with suchrecombinant expression vectors, and processes for the manufacture of therecombinant human factor VIII and its derivatives. The invention alsocovers a transfer vector for use in human gene therapy which comprisessuch modified DNA sequences.

[0002] Classic hemophilia or hemophilia A is the most common of theinherited bleeding disorders. It results from a chromosome X-linkeddeficiency of blood coagulation factor VIII, and affects almostexclusively males with an incidence of between one and two individualsper 10.000. The X-chromosome defect is transmitted by female carrierswho are not themselves hemophiliacs. The clinical manifestation ofhemophilia A is an abnormal bleeding tendency and before treatment withfactor VIII concentrates was introduced the mean life span for a personwith severe hemophilia was less than 20 years. The use of concentratesof factor VIII from plasma has considerably improved the situation forthe hemophilia patients. The mean life span has increased extensively,giving most of them the possibility to live a more or less normal life.However, there have been certain problems with the plasma derivedconcentrates and their use, the most serious of which have been thetransmission of viruses. So far, viruses causing AIDS, hepatitis B, andnon A non B hepatitis have hit the population seriously. Althoughdifferent virus inactivation methods and new highly purified factor VIIIconcentrates have recently been developed an inadvertant contaminationcan not be excluded. Also, the factor VIII concentrates are fairlyexpensive because of the limited supply of human plasma raw material.

[0003] A factor VIII product derived from recombinant material is likelyto solve a large extent of the problems associated with the use ofplasma derived factor VIII concentrates for treatment for hemophilia A.However, the development of a recombinant factor VIII has met somedifficulties, for instance the problem of achieving production levels insufficiently high yields, in particular regarding the full-lengthmolecule.

[0004] In fresh plasma prepared in the presence of protease inhibitors,factor VIII has been shown to have a molecular weight of 280 kDa and tobe composed of two polypeptide chains of 200 kDa and 80 kDa,respectively (Andersson, L.-O., et al. (1986) Proc. Natl. Acad. Sci. USA83, 2979-2983). These chains are held together by metal ion bridges.More or less proteolytically degraded forms of the factor VIII moleculecan be found as active fragments in factor VIII material purified fromcommercial concentrates (Andersson, L.-O., et al. ibid.; Andersson,L.-O., et al. (1985) EP 0 197 901). The fragmented form of factor VIIIhaving molecular weights from 260 kDa down to 170 kDa, consists of oneheavy chain with a molecular weight ranging from 180 kDa down to 90 kDa,where all variants have identical amino termini, in combination with one80 kDa light chain. The amino-terminal region of the heavy chain isidentical to that of the single chain factor VIII polypeptide that canbe deduced from the nucleotide sequence data of the factor VIII cDNA(Wood, W. I., et al. (1984) Nature 312, 330-336; Vehar, G. A., et al.(1984) Nature 312, 337-342).

[0005] The smallest active form of factor VIII with a molecular weightof 170 kDa, consisting of one 90 kDa and one 80 kDa chain, can beactivated with thrombin to the same extent as the higher molecularweight forms, and thus represents an unactivated form. It has also beenshown to have full biological activity in vivo as tested in hemophiliadogs (Brinkhous, K. M., et al. (1985) Proc. Natl. Acad. Sci. USA 82,8752-8756). Thus, the haemostatic effectiveness of the 170 kDa form isthe same as for the high molecular weight forms of factor VIII.

[0006] The fact that the middle heavily glycosylated region of thefactor VIII polypeptide chain residing between amino acids Arg-740 andGlu-1649 does not seem to be necessary for full biological activity hasprompted several researchers to attempt to produce derivatives ofrecombinant factor VIII lacking this region. This has been achieved bydeleting a portion of the cDNA encoding the middle heavily glycosylatedregion of factor VIII either entirely or partially.

[0007] For example, J. J. Toole, et al, reported the construction andexpression of factor VIII lacking amino acids 982 through 1562, and 760through 1639 respectively (Proc. Natl. Acad. Sci. USA (1986) 83,5939-5942). D. L. Eaton, et al. reported the construction and expressionof factor VIII lacking amino acids 797 through 1562 (Biochemistry (1986)25, 8343-8347). R. J. Kaufman described the expression of factor VIIIlacking amino acids 741 through 1646 (PCT application No. WO 87/04187).N. Sarver, et al. reported the construction and expression of factorVIII lacking amino acids 747 through 1560 (DNA (1987) 6, 553-564). M.Pasek reported the construction and expression of factor VIII lackingamino acids 745 through 1562, and amino acids 741 through 1648,respectively (PCT application No. WO 88/00831). K.-D. Langner reportedthe construction and expression of factor VIII lacking amino acids 816through 1598, and amino acids 741 through 1689, respectively (BehringInst. Mitt., (1988) No. 82, 16-25, EP 0 295 597). P. Meulien, et al.,reported the construction and expression of factor VIII lacking aminoacids 868 through 1562, and amino acids 771 through 1666, respectively(Protein Engineering (1988) 2(4), 301-306, EP 0 303 540). Whenexpressing these deleted forms of factor VIII cDNA in mammalian cellsthe production level is typically 10 times higher as compared tofull-length factor VIII.

[0008] FVIII is secreted into plasma as a heterodimer of a heavy chain(domains A1-A2-B) and a light chain (A3-C1-C2) associated through anoncovalent divalent metal ion linkage between the A1- and A3 domains.In plasma, FVIII is stabilized by binding to von Willebrand factor.

[0009] Upon proteolytic activation by thrombin, FVIII is activated to aheterotrimer of 2 heavy chain fragments (A1, a 50 kDa fragment, and A2 a43 kDa fragment) and the light chain (A3-C1-C2, a 73 kDa fragment). Theactive form of FVIII (FVIIIa) thus consists of an A1-subunit associatedthrough the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2light chain and a free A2 subunit associated with the A1 domain. Thedissociation of that free A2 subunit from the heterotrimer is thought tobe the rate limiting step in FVIIIa inactivation after thrombinactivation (Fay, P. J. et al, J. Biol. Chem. 266: 8957 (1991), Fay P J &Smudzin T M, J. Biol. Chem. 267: 13246-50 (1992)). The half life ofFVIIIa in plasma is only 2.1 minutes (Saenko et al., Vox Sang. 83: 89-96(2002)). To enhance the half life of FVIIIa would result into a longeracting FVIIIa which would also translate into less frequent injectionsof such a FVIII preparation. The inactivation of FVIIIa throughactivated Protein C (APC) by cleavage at Arg336 and Arg562 is thoughtnot to be the rate limiting step. Attempts have been made to create aFVIIIa which is inactivation resistant by covalently attaching the A2domain to the A3 domain and by mutating the APC cleavage sites (Pipe andKaufman, PNAS, 94:11851-11856). However such a FVIIIa could have athrombogenic potential as it is almost completely inactivationresistant. It is therefore the purpose of this invention to create aFVIIIa in which the A2 domain is stabilized without completely blockinginactivation.

[0010] FVIII is administered i.v. to haemophilia patients who are onprophylactic treatment about 3 times per week due to the plasma halflife of FVIII of about 12 hours. It would thus be highly desirable tocreate a FVIII with enhanced plasma half life which could lead to aFVIII preparation which has to be administered less frequently. Thepresent invention offers a solution to this problem by a modified FVIIImolecule with an increased association of A2 to the A1/A3-C1-C2.

[0011] The nature of these modifications was identified by comparing thesequence of porcine FVIII to that of human FVIII as it is known that thedissociation of human A2 domain is threefold enhanced versus that ofporcine A2 (Lollar et al., J Biol. Chem., 267:23652-23657 (1992)). Thesequence comparison (FIG. 1) revealed several differences. A subset ofthese differences consists of differently charged amino acids. Mutantsof human FVIII were constructed according to the following guidelines.When the human sequence contained a neutral amino acid whereas theporcine sequence contained a charged amino acid then a charged aminoacid with the same charge as found in the porcine sequence wasintroduced into the human sequence. When the human sequence contained acharged amino acid whereas the porcine FVIII contained a neutral aminoacid then a neutral amino acid or an amino acid of the opposite chargewas introduced, e.g. if the human FVIII contained an acidic amino acidat a position where the porcine FVIII contained a neutral amino acid,also a basic amino acid was introduced. When the human sequencecontained a charged sequence whereas the porcine FVIII contained acharged amino acid then an amino acid with the same charge as found inthe porcine amino acid was introduced into the human sequence. Examplesfor such mutations which lead to an improved FVIII with a plasma halflife of its activated form of more than three minutes, preferably ofmore than 10 minutes even more preferably more than 30 minutes, arelisted in FIG. 2.

[0012] Other mutations for an improved FVIII were deduced by analyzingmutations in human FVIII which occurred naturally and which lead to afaster dissociation of the A2 domain associated with hemophilia. Suchmutations result in differences between the two-stage assay as comparedto the one-stage assay while determining FVIII clotting activity,whereas the two-stage assay result is lower than that of the one-stageassay as in the two-stage assay an incubation time of several minutesallows an unstable A2 domain to dissociate (Saenko et al., Vox Sang.,83:89-96 (2002). It was inferred that in those cases where such anincreased instability was the result of the introduction of a chargedamino acid that amino acid should be mutated into one of the oppositecharge. Examples for such mutations which lead to an improved FVIII witha plasma half life of its activated form of more than three minutes,preferably of more than 10 minutes, even more preferably more than 30minutes, are listed in FIG. 3.

[0013] As a basis for introducing the mutations preferably a modifiedfactor VIII cDNA is used which comprises a first DNA segment coding forthe amino acids 1 through 740 of the human factor VIII and a second DNAsegment coding for the amino acids 1649 through 2332 of the human factorVIII. These two segments may be interconnected by a linker DNA segment,but the invention also encompasses introducing the mutations into fulllength FVIII.

[0014] Subject of the invention is therefore a modified human factorVIII cDNA wherein mutations are inserted either in the wild-type factorVIII cDNA or in a factor VIII cDNA in which the B-domain is partially orcompletely deleted and may be replaced by a DNA linker segment, and

[0015] A) one or several codons of the human factor VIII cDNA which arenot identical with the corresponding codon in the same position of theporcine factor VIII cDNA are substituted by a different codon in such away that

[0016] when the human sequence contains a codon for a neutral amino acidwhereas the porcine sequence contains a codon for a charged amino acidthen a codon for an amino acid with the same charge as found in theporcine sequence is introduced into the human sequence;

[0017] when the human sequence contains a codon for a charged amino acidwhereas the porcine sequence contains a codon for a neutral amino acidthen a codon for a neutral amino acid or a codon for an amino acid ofthe opposite charge is introduced into the human sequence

[0018] when the human sequence contains a codon for a charged amino acidwhereas the porcine sequence contains a codon for an amino acid with theopposite charge then a codon for an amino acid with the opposite chargeis introduced into the human sequence or

[0019] B) one or several codons for a charged amino acid which are foundin the FVIII cDNA of a hemophilic patient are replaced by a codon for anamino acid of the opposite charge.

[0020] The production of factor VIII proteins at high levels in suitablehost cells, requires the assembly of the above-mentioned modified factorVIII DNA's into efficient transcriptional units together with suitableregulatory elements in a recombinant expression vector, that can bepropagated in E. coli according to methods known to those skilled in theart. Efficient transcriptional regulatory elements could be derived fromviruses having animal cells as their natural hosts or from thechromosomal DNA of animal cells. Preferably, promoter-enhancercombinations derived from the Simian Virus 40, adenovirus, BK polyomavirus, human cytomegalovirus, or the long terminal repeat of Roussarcoma virus, or promoter-enhancer combinations including stronglyconstitutively transcribed genes in animal cells like beta-actin orGRP78 can be used. In order to achieve stable high levels of mRNAtranscribed from the factor VIII DNA's, the transcriptional unit shouldcontain in its 3′-proximal part a DNA region encoding a transcriptionaltermination-polyadenylation sequence. Preferably, this sequence isderived from the Simian Virus 40 early transcriptional region, therabbit beta-globin gene, or the human tissue plasminogen activator gene.

[0021] The factor VIII cDNA's are then integrated into the genome into asuitable host cell line for expression of the factor VIII proteins.Preferably this cell line should be an animal cell-line of vertebrateorigin in order to ensure correct folding, disulfide bond formation,asparagines-linked glycosylation and other post-translationalmodifications as well as secretion into the cultivation medium. Exampleson other post-translational modifications are tyrosine O-sulfation, andproteolytic processing of the nascent polypeptide chain. Examples ofcell lines that can be use are monkey COS-cells, mouse L-cells, mouseC127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells, andpreferentially CHO-cells.

[0022] The recombinant expression vector encoding the factor VIII cDNA'scan be introduced into an animal cell line in several different ways.For instance, recombinant expression vectors can be created from vectorsbased on different animal viruses, Examples of these are vectors basedon baculovirus, vaccinia virus, adenovirus, and preferably bovinepapilloma virus.

[0023] The transcription units encoding the factor VIII DNA's can alsobe introduced into animal cells together with another recombinant genewhich may function as a dominant selectable marker in these cells inorder to facilitate the isolation of specific cell clones which haveintegrated the recombinant DNA into their genome. Examples of this typeof dominant selectable marker genes are Tn5 aminoglycosidephosphotransferase, conferring resistance to Geneticin (G418),hygromycin phosphotransferase, conferring resistance to hygromycin, andpuromycin acetyl transferase, conferring resistance to puromycin. Therecombinant expression vector encoding such a selectable marker canreside either on the same vector as the one encoding the factor VIIIcDNA, or it can be encoded on a separate vector which is simultaneouslyintroduced and integrated to the genome of the host cell, frequentlyresulting in a tight physical linkage between the differenttranscription units.

[0024] Other types of selectable marker genes which can be used togetherwith the factor VIII DNA's are based on various transcription unitsencoding dihydrofolate reductase (dhfr). After introduction of this typeof gene into cells lacking endogenous dhfr-activity, preferentiallyCHO-cells (DUKX-B11, DG-44) it will enable these to grow in medialacking nucleosides. An example of such a medium is Ham's F12 withouthypoxanthin, thymidin, and glycine. These dhfr-genes can be introducedtogether with the factor VIII cDNA transcriptional units into CHO-cellsof the above type, either linked on the same vector or on differentvectors, thus creating dhfr-positive cell lines producing recombinantfactor VIII protein.

[0025] If the above cell lines are grown in the presence of thecytotoxic dhfr-inhibitor methotrexate, new cell lines resistant tomethotrexate will emerge. These cell lines may produce recombinantfactor VIII protein at an increased rate due to the amplified number oflinked dhfr and factor VIII transcriptional units. When propagatingthese cell lines in increasing concentrations of methotrexate (1-10000nM), new cell lines can be obtained which produce factor VIII protein atvery high rate.

[0026] The above cell lines producing factor VIII protein can be grownon a large scale, either in suspension culture or on various solidsupports. Examples of these supports are microcarriers based on dextranor collagen matrices, or solid supports in the form of hollow fibres orvarious ceramic materials. When grown in cell suspension culture or onmicrocarriers the culture of the above cell lines can be performedeither as a bath culture or as a perfusion culture with continuousproduction of conditioned medium over extended periods of time. Thus,according to the present invention, the above cell lines are well suitedfor the development of an industrial process for the production ofrecombinant factor VIII that can be isolated from human plasma.

[0027] The recombinant factor VIII protein which accumulate in themedium of CHO-cells of the above type, can be concentrated and purifiedby a variety of biochemical and chromatographic methods, includingmethods utilizing differences in size, charge, hydrophobicity,solubility, specific affinity, etc. between the recombinant factor VIIIprotein and other substances in the cell cultivation medium.

[0028] An example of such a purification is the adsorption of therecombinant factor VIII protein to a monoclonal antibody which isimmobilised on a solid support. After desorption, the factor VIIIprotein can be further purified by a variety of chromatographictechniques based on the above properties.

[0029] The recombinant proteins with factor VIII activity described inthis invention can be formulated into pharmaceutical preparations fortherapeutic use. The purified factor VIII proteins may be dissolved inconventional physiologically compatible aqueous buffer solutions towhich there may be added, optionally, pharmaceutical adjuvants toprovide pharmaceutical preparations.

[0030] The modified factor VIII DNA's of this invention may also beintegrated into a transfer vector for use in the human gene therapy.

[0031] A further subject of this invention is a modified biologicallyactive recombinant human factor VIII with improved plasma half life ofits activated form wherein mutations are inserted either in thewild-type factor VIII or in a FVIII in which the B-domain is partiallyor completely deleted and replaced by a linker, and

[0032] A) one or several amino acids of the human factor VIII which arenot identical with the corresponding amino acid in the same position ofthe porcine factor VIII are substituted by a different amino acid insuch a way that

[0033] when the human sequence contains a neutral amino acid whereas theporcine sequence contains a charged amino acid then a charged amino acidwith the same charge as found in the porcine sequence is introduced intothe human sequence;

[0034] when the human sequence contains a charged amino acid whereas theporcine sequence contains a neutral amino acid then a neutral amino acidor an amino acid of the opposite charge is introduced into the humansequence;

[0035] when the human sequence contains a charged amino acid whereas theporcine sequence contains an amino acid with the opposite charge then anamino acid with the opposite charge is introduced into the humansequence or

[0036] B) one or several charged amino acids which are found in theFVIII amino sequence of hemophilic patients are replaced by a codon foran amino acid of the opposite charge.

[0037] The present invention will be further described more in detail inthe following examples thereof. This description of specific embodimentsof the invention will be made in conjunction with the appended figures.

[0038] Generation of FVIII mutants

[0039] For the generation of FVIII mutants, a suitable subfragment ofthe FVIII cDNA (e.g. Aval—Sacl, encompassing aminoacids 226 to 978) isfirst subcloned into a suitable cloning vector to reduce subsequentsequencing efforts. Site directed mutagenesis is then performed with acommercially available mutagenesis kit (e.g. QuickChange SiteDirectedMutagenesis Kit (Stratagene) according to the manufacturer'sinstructions. Primers used for mutagenesis are listed in the attachedsequence listing and below, where the mutagenic bases are indicated inbold letters. Mutation: A284K Forward primer^(5′)GGAACCATCGCCAGAAGTCCTTGGAAATCTCGCC^(3′) (Sequence 1) Reverse primer5′GGCGAGATTTCCAAGGACTTCTGGCGATGGTTCC^(3′) (Sequence 2) Mutation: D318GForward primer ^(5′)CCCACCAACATGGTGGCATGGAAGCTTATGTC^(3′) (Sequence 3)Reverse primer ^(5′)GACATAAGCTTCCATGCCACCATGTTGGTGGG^(3′) (Sequence 4)Mutation: M337R Forward primer^(5′)CAGAGGAACCCCAACTACGACGTAAAAATAATGAAGAAGCGGAAGAC^(3′) (Sequence 5)Reverse primer ^(5′)GTCTTCCGCTTCTTCATTATTTTTACGTCGTAGTTGGGGTTCCTCTG^(3′)(Sequence 6) Mutation: N340D Forward primer^(5′)CCCAACTACGAATGAAAAATGATGAAGAAGCGGAAGACTATG^(3′) (Sequence 7)Reverse primer ^(5′)CATAGTCTTCCGCTTCTTCATCATTTTTCATTCGTAGTTGGG^(3′)(Sequence 8) Mutation: D349N Forward primer^(5′)GAAGAAGCGGAAGACTATGATGATAATCTTACTGATTCTG^(3′) (Sequence 9) Reverseprimer ^(5′)CAGAATCAGTAAGATTATCATCATAGTCTTCCGCTTCTTC^(3′) (Sequence 10)Mutation: N364D Forward primer^(5′)GGTCAGGTTTGATGATGACGACTCTCCTTCCTTTATCC^(3′) (Sequence 11) Reverseprimer ^(5′)GGATAAAGGAAGGAGAGTCGTCATCATCAAACCTGACC^(3′) (Sequence 12)Mutation: D403S Forward primer^(5′)CCCTTAGTCCTCGCCCCCTCTGACAGAAGTTATAAAAG^(3′) (Sequence 13) Reverseprimer ^(5′)CTTTTATAACTTCTGTCAGAGGGGGCGAGGACTAAGGG^(3′) (Sequence 14)Mutation: E434V Forward primer^(5′)GTCCGATTTATGGCATACACAGATGTTACCTTTAAGACTCG^(3′) (Sequence 15)Reverse primer ^(5′)CGAGTCTTAAAGGTAACATCTGTGTATGCCATAAATCGGAC^(3′)(Sequence 16) Mutation: E440K Forward primer^(5′)CCTTTAAGACTCGTAAAGCTATTCAGCATGAATCAGG^(3′) (Sequence 17) Reverseprimer ^(5′)CCTGATTCATGCTGAATAGCTTTACGAGTCTTAAAGG^(3′) (Sequence 18)Mutation: Q468K Forward primer^(5′)CACACTGTTGATTATATTTAAGAATAAAGCAAGCAGACCATATAAC^(3′) (Sequence 19)Reverse primer ^(5′)GTTATATGGTCTGCTTGCTTTATTCTTAAATATAATCAACAGTGTG^(3′)(Sequence 20) Mutation: R484S Forward primer^(5′)CCCTCACGGAATCACTGATGTCTCTCCTTTGTATTCAAGG^(3′) (Sequence 21) Reverseprimer ^(5′)CCTTGAATACAAAGGAGAGACATCAGTGATTCCGTGAGGG^(3′) (Sequence 22)Mutation: R489G Forward primer^(5′)GATGTCCGTCCTTTGTATTCAGGGAGATTACCAAAAGG^(3′) (Sequence 23) Reverseprimer ^(5′)CCTTTTGGTAATCTCCCTGAATACAAAGGACGGACAT^(3′) (Sequence 24)Mutation: R583Q Forward primer^(5′)CTGTATTTGATGAGAACCAAAGCTGGTACCTCACAG^(3′) (Sequence 25) Reverseprimer ^(5′)CTGTGAGGTACCAGCTTTGGTTCTCATCAAATAGAG^(3′) (Sequence 26)Mutation: A599D Forward primer ^(5′)CTCCCCAATCCAGATGGAGTGCAGCTTGAG^(3′)(Sequence 27) Reverse primer ^(5′)CTCAAGCTGCACTCCATCTGGATTGGGGAG^(3′)(Sequence 28) Mutation: E604Q Forward primer^(5′)CAGCTGGAGTGCAGCTTCAGGATCCAGAGTTC^(3′) (Sequence 29) Reverse primer^(5′)GAACTCTGGATCCTGAAGCTGCACTCCAGCTG^(3′) (Sequence 30)Mutation: G1948K Forward primer^(5′)CGATGGTATCTGCTCAGCATGAAGAGCAATGAAAACATCCATTCTATTC^(3′) (Sequence31) Reverse primer^(5′)GAATAGAATGGATGTTTTCATTGCTCTTCATGCTGAGCAGATACCATCG^(3′) (Sequence32)

[0040] After clone isolation and sequence verification mutantsubfragments are reinserted into the respective expression vector.

[0041] Expression of FVIII Mutants

[0042] Transfection of FVIII mutant clones and expression of the mutantFVIII molecules is done as described previously and known to thoseskilled in the art (e.g. Plantier J L et al. Thromb. Haemost. 86:596-603(2001)).

[0043] Measuring Affinity of A2 Subunit for A1/A3-C1-C2

[0044] The increased affinity of the A2 subunit for the A1/A3-C1-C2 canbe measured as previously described by functional assays (Fay P J &Smudzin T M. J. Biol. Chem. 267:13246-50 (1992); Lollar P et al. J.Biol. Chem. 267:23652-57 (1992)) as well as a physical assay employingsurface plasmon resonance (Persson E et al. Biochemistry 34:12775-81(1995)).

[0045] The sequence of the porcine factor VIII is shown in Sequence 33,whereas the sequence of the human factor VIII is shown in sequence 34 ofthe attached sequence listing.

[0046] In the following Sequence Listing Sequences 1-32 describeoligonucleotides which are used to introduce specific mutations intoFVIII. Sequence 33 is the amino acid sequence of full length matureporcine FVIII, Sequence 34 is the amino acid sequence of full lengthmature human FVIII.

1 34 1 34 DNA Artificial Sequence Human Factor VIII derivatives 1ggaaccatcg ccagaagtcc ttggaaatct cgcc 34 2 34 DNA Artificial SequenceHuman Factor VIII derivatives 2 ggcgagattt ccaaggactt ctggcgatgg ttcc 343 32 DNA Artificial Sequence Human Factor VIII derivatives 3 cccaccaacatggtggcatg gaagcttatg tc 32 4 32 DNA Artificial Sequence Human FactorVIII derivatives 4 gacataagct tccatgccac catgttggtg gg 32 5 47 DNAArtificial Sequence Human Factor VIII derivatives 5 cagaggaaccccaactacga cgtaaaaata atgaagaagc ggaagac 47 6 47 DNA Artificial SequenceHuman Factor VIII derivatives 6 gtcttccgct tcttcattat ttttacgtcgtagttggggt tcctctg 47 7 42 DNA Artificial Sequence Human Factor VIIIderivatives 7 cccaactacg aatgaaaaat gatgaagaag cggaagacta tg 42 8 42 DNAArtificial Sequence Human Factor VIII derivatives 8 catagtcttccgcttcttca tcatttttca ttcgtagttg gg 42 9 40 DNA Artificial SequenceHuman Factor VIII derivatives 9 gaagaagcgg aagactatga tgataatcttactgattctg 40 10 40 DNA Artificial Sequence Human Factor VIIIderivatives 10 cagaatcagt aagattatca tcatagtctt ccgcttcttc 40 11 38 DNAArtificial Sequence Human Factor VIII derivatives 11 ggtcaggtttgatgatgacg actctccttc ctttatcc 38 12 38 DNA Artificial Sequence HumanFactor VIII derivatives 12 ggataaagga aggagagtcg tcatcatcaa acctgacc 3813 38 DNA Artificial Sequence Human Factor VIII derivatives 13cccttagtcc tcgccccctc tgacagaagt tataaaag 38 14 38 DNA ArtificialSequence Human Factor VIII derivatives 14 cttttataac ttctgtcagagggggcgagg actaaggg 38 15 41 DNA Artificial Sequence Human Factor VIIIderivatives 15 gtccgattta tggcatacac agatgttacc tttaagactc g 41 16 41DNA Artificial Sequence Human Factor VIII derivatives 16 cgagtcttaaaggtaacatc tgtgtatgcc ataaatcgga c 41 17 37 DNA Artificial SequenceHuman Factor VIII derivatives 17 cctttaagac tcgtaaagct attcagcatgaatcagg 37 18 37 DNA Artificial Sequence Human Factor VIII derivatives18 cctgattcat gctgaatagc tttacgagtc ttaaagg 37 19 46 DNA ArtificialSequence Human Factor VIII derivatives 19 cacactgttg attatatttaagaataaagc aagcagacca tataac 46 20 46 DNA Artificial Sequence HumanFactor VIII derivatives 20 gttatatggt ctgcttgctt tattcttaaa tataatcaacagtgtg 46 21 40 DNA Artificial Sequence Human Factor VIII derivatives 21ccctcacgga atcactgatg tctctccttt gtattcaagg 40 22 40 DNA ArtificialSequence Human Factor VIII derivatives 22 ccttgaatac aaaggagagacatcagtgat tccgtgaggg 40 23 38 DNA Artificial Sequence Human Factor VIIIderivatives 23 gatgtccgtc ctttgtattc agggagatta ccaaaagg 38 24 38 DNAArtificial Sequence Human Factor VIII derivatives 24 ccttttggtaatctccctga atacaaagga cggacatc 38 25 36 DNA Artificial Sequence HumanFactor VIII derivatives 25 ctgtatttga tgagaaccaa agctggtacc tcacag 36 2636 DNA Artificial Sequence Human Factor VIII derivatives 26 ctgtgaggtaccagctttgg ttctcatcaa atacag 36 27 30 DNA Artificial Sequence HumanFactor VIII derivatives 27 ctccccaatc cagatggagt gcagcttgag 30 28 30 DNAArtificial Sequence Human Factor VIII derivatives 28 ctcaagctgcactccatctg gattggggag 30 29 32 DNA Artificial Sequence Human Factor VIIIderivatives 29 cagctggagt gcagcttcag gatccagagt tc 32 30 32 DNAArtificial Sequence Human Factor VIII derivatives 30 gaactctggatcctgaagct gcactccagc tg 32 31 49 DNA Artificial Sequence Human FactorVIII derivatives 31 cgatggtatc tgctcagcat gaagagcaat gaaaacatccattctattc 49 32 49 DNA Artificial Sequence Human Factor VIII derivatives32 gaatagaatg gatgttttca ttgctcttca tgctgagcag ataccatcg 49 33 2114 PRTPorcine 33 Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp AspTyr 1 5 10 15 Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr ArgPhe Pro 20 25 30 Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val LeuTyr Lys 35 40 45 Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser ValAla Arg 50 55 60 Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile GlnAla Glu 65 70 75 80 Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met AlaSer His Pro 85 90 95 Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys SerSer Glu Gly 100 105 110 Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu LysGlu Asp Asp Lys 115 120 125 Val Leu Pro Gly Lys Ser Gln Thr Tyr Val TrpGln Val Leu Lys Glu 130 135 140 Asn Gly Pro Thr Ala Ser Asp Pro Pro CysLeu Thr Tyr Ser Tyr Leu 145 150 155 160 Ser His Val Asp Leu Val Lys AspLeu Asn Ser Gly Leu Ile Gly Ala 165 170 175 Leu Leu Val Cys Arg Glu GlySer Leu Thr Arg Glu Arg Thr Gln Asn 180 185 190 Leu His Glu Phe Val LeuLeu Phe Ala Val Phe Asp Glu Gly Lys Ser 195 200 205 Trp His Ser Ala ArgAsn Asp Ser Trp Thr Arg Ala Met Asp Pro Ala 210 215 220 Pro Ala Arg AlaGln Pro Ala Met His Thr Val Asn Gly Tyr Val Asn 225 230 235 240 Arg SerLeu Pro Gly Leu Ile Gly Cys His Lys Lys Ser Val Tyr Trp 245 250 255 HisVal Ile Gly Met Gly Thr Ser Pro Glu Val His Ser Ile Phe Leu 260 265 270Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala Ser Leu Glu 275 280285 Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu Met Asp Leu 290295 300 Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His His Gly Gly305 310 315 320 Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu ProGln Leu 325 330 335 Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp AsnLeu Tyr Asp 340 345 350 Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp AspVal Ser Pro Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His ProLys Thr Trp Val His 370 375 380 Tyr Ile Ser Ala Glu Glu Glu Asp Trp AspTyr Ala Pro Ala Val Pro 385 390 395 400 Ser Pro Ser Asp Arg Ser Tyr LysSer Leu Tyr Leu Asn Ser Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys TyrLys Lys Ala Arg Phe Val Ala Tyr Thr 420 425 430 Asp Val Thr Phe Lys ThrArg Lys Ala Ile Pro Tyr Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu LeuTyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn LysAla Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr AspVal Ser Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys 485 490 495 HisLeu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520525 Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys Asp Leu Ala 530535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560 Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val IleLeu Phe 565 570 575 Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala GluAsn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro GlnAsp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn GlyTyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu ValAla Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Val Gly Ala Gln Thr Asp PheLeu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met ValTyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr ValPhe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Val Leu Gly Cys HisAsn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys ValTyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp 705 710 715 720 Asn ThrTyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val 725 730 735 IleGlu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser 740 745 750Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp Val Glu Leu 755 760765 Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu Leu Ser Val 770775 780 Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro Ala Pro His785 790 795 800 Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu AlaAsp Asp 805 810 815 Tyr Leu Pro Gly Ala Arg Glu Arg Asn Thr Ala Pro SerAla Ala Ala 820 825 830 Arg Leu Arg Pro Glu Leu His His Ser Ala Glu ArgVal Leu Thr Pro 835 840 845 Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp SerLys Met Ser Ser Ser 850 855 860 Ser Asp Leu Leu Lys Thr Ser Pro Thr IlePro Ser Asp Thr Leu Ser 865 870 875 880 Ala Glu Thr Glu Arg Thr His SerLeu Gly Pro Pro His Pro Gln Val 885 890 895 Asn Phe Arg Ser Gln Leu GlyAla Ile Val Leu Gly Lys Asn Ser Ser 900 905 910 His Phe Ile Gly Ala GlyVal Pro Leu Gly Ser Thr Glu Glu Asp His 915 920 925 Glu Ser Ser Leu GlyGlu Asn Val Ser Pro Val Glu Ser Asp Gly Ile 930 935 940 Phe Glu Lys GluArg Ala His Gly Pro Ala Ser Leu Thr Lys Asp Asp 945 950 955 960 Val LeuPhe Lys Val Asn Ile Ser Leu Val Lys Thr Asn Lys Ala Arg 965 970 975 ValTyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp Ala Ala Leu 980 985 990Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys Asn Thr Thr 995 10001005 Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly Pro Leu 10101015 1020 Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu Leu1025 1030 1035 Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln SerSer 1040 1045 1050 Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu GluLeu Ser 1055 1060 1065 Lys Gly Lys Glu Met Met Leu Pro Asn Ser Glu LeuThr Phe Leu 1070 1075 1080 Thr Asn Ser Ala Asp Val Gln Gly Asn Asp ThrHis Ser Gln Gly 1085 1090 1095 Lys Lys Ser Arg Glu Glu Met Glu Arg ArgGlu Lys Leu Val Gln 1100 1105 1110 Glu Lys Val Asp Leu Pro Gln Val TyrThr Ala Thr Gly Thr Lys 1115 1120 1125 Asn Phe Leu Arg Asn Ile Phe HisGln Ser Thr Glu Pro Ser Val 1130 1135 1140 Glu Gly Phe Asp Gly Gly SerHis Ala Pro Val Pro Gln Asp Ser 1145 1150 1155 Arg Ser Leu Asn Asp SerAla Glu Arg Ala Glu Thr His Ile Ala 1160 1165 1170 His Phe Ser Ala IleArg Glu Glu Ala Pro Leu Glu Ala Pro Gly 1175 1180 1185 Asn Arg Thr GlyPro Gly Pro Arg Ser Ala Val Pro Arg Arg Val 1190 1195 1200 Lys Gln SerLeu Lys Gln Ile Arg Leu Pro Leu Glu Glu Ile Lys 1205 1210 1215 Pro GluArg Gly Val Val Leu Asn Ala Thr Ser Thr Arg Trp Ser 1220 1225 1230 GluSer Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn Asn Leu Ser 1235 1240 1245Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly Lys Ile 1250 12551260 Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser Gly Lys 12651270 1275 Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser1280 1285 1290 Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg GluAsp 1295 1300 1305 Leu Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala HisGly Asp 1310 1315 1320 Leu Gly Gln Glu Ile Phe Leu Gln Lys Thr Arg GlyPro Val Asn 1325 1330 1335 Leu Asn Lys Val Asn Arg Pro Gly Arg Thr ProSer Lys Leu Leu 1340 1345 1350 Gly Pro Pro Met Pro Lys Glu Trp Glu SerLeu Glu Lys Ser Pro 1355 1360 1365 Lys Ser Thr Ala Leu Arg Thr Lys AspIle Ile Ser Leu Pro Leu 1370 1375 1380 Asp Arg His Glu Ser Asn His SerIle Ala Ala Lys Asn Glu Gly 1385 1390 1395 Gln Ala Glu Thr Gln Arg GluAla Ala Trp Thr Lys Gln Gly Gly 1400 1405 1410 Pro Gly Arg Leu Cys AlaPro Lys Pro Pro Val Leu Arg Arg His 1415 1420 1425 Gln Arg Asp Ile SerLeu Pro Thr Phe Gln Pro Glu Glu Asp Lys 1430 1435 1440 Met Asp Tyr AspAsp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp 1445 1450 1455 Phe Asp IleTyr Gly Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe 1460 1465 1470 Gln LysArg Thr Arg His Tyr Phe Ile Ala Ala Val Glu Gln Leu 1475 1480 1485 TrpAsp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg Asn Arg 1490 1495 1500Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe Arg 1505 15101515 Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu 15201525 1530 Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu1535 1540 1545 Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala SerArg 1550 1555 1560 Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro AspAsp Gln 1565 1570 1575 Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val GlnPro Asn Glu 1580 1585 1590 Thr Arg Thr Tyr Phe Trp Lys Val Gln His HisMet Ala Pro Thr 1595 1600 1605 Glu Asp Glu Phe Asp Cys Lys Ala Trp AlaTyr Phe Ser Asp Val 1610 1615 1620 Asp Leu Glu Lys Asp Val His Ser GlyLeu Ile Gly Pro Leu Leu 1625 1630 1635 Ile Cys Arg Ala Asn Thr Leu AsnAla Ala His Gly Arg Gln Val 1640 1645 1650 Thr Val Gln Glu Phe Ala LeuPhe Phe Thr Ile Phe Asp Glu Thr 1655 1660 1665 Lys Ser Trp Tyr Phe ThrGlu Asn Val Glu Arg Asn Cys Arg Ala 1670 1675 1680 Pro Cys His Leu GlnMet Glu Asp Pro Thr Leu Lys Glu Asn Tyr 1685 1690 1695 Arg Phe His AlaIle Asn Gly Tyr Val Met Asp Thr Leu Pro Gly 1700 1705 1710 Leu Val MetAla Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser 1715 1720 1725 Met GlySer Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His 1730 1735 1740 ValPhe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr 1745 1750 1755Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser 1760 17651770 Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu 17751780 1785 Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys1790 1795 1800 Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp PheGln 1805 1810 1815 Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro LysLeu Ala 1820 1825 1830 Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp SerThr Lys Asp 1835 1840 1845 Pro His Ser Trp Ile Lys Val Asp Leu Leu AlaPro Met Ile Ile 1850 1855 1860 His Gly Ile Met Thr Gln Gly Ala Arg GlnLys Phe Ser Ser Leu 1865 1870 1875 Tyr Ile Ser Gln Phe Ile Ile Met TyrSer Leu Asp Gly Arg Asn 1880 1885 1890 Trp Gln Ser Tyr Arg Gly Asn SerThr Gly Thr Leu Met Val Phe 1895 1900 1905 Phe Gly Asn Val Asp Ala SerGly Ile Lys His Asn Ile Phe Asn 1910 1915 1920 Pro Pro Ile Val Ala ArgTyr Ile Arg Leu His Pro Thr His Tyr 1925 1930 1935 Ser Ile Arg Ser ThrLeu Arg Met Glu Leu Met Gly Cys Asp Leu 1940 1945 1950 Asn Ser Cys SerMet Pro Leu Gly Met Gln Asn Lys Ala Ile Ser 1955 1960 1965 Asp Ser GlnIle Thr Ala Ser Ser His Leu Ser Asn Ile Phe Ala 1970 1975 1980 Thr TrpSer Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr 1985 1990 1995 AsnAla Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln 2000 2005 2010Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln 2015 20202025 Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu 20302035 2040 Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln2045 2050 2055 Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser SerThr 2060 2065 2070 Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr ArgTyr Leu 2075 2080 2085 Arg Ile His Pro Thr Ser Trp Ala Gln His Ile AlaLeu Arg Leu 2090 2095 2100 Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr2105 2110 34 2332 PRT Homo sapiens 34 Ala Thr Arg Arg Tyr Tyr Leu GlyAla Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly GluLeu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe ProPhe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe ThrAsp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly LeuLeu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val IleThr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val GlyVal Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp GlnThr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly GlySer His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro MetAla Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 HisVal Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val AsnArg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser ValTyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His SerIle Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg GlnAla Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln ThrLeu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile SerSer His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val AspSer Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu GluAla Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp ValVal Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile ArgSer Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile AlaAla Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 AlaPro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His GlyIle 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro LysGly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu IlePhe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr LysSer Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val AsnMet Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu IleCys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile MetSer Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu AsnArg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro AsnPro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser AsnIle Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln LeuSer Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 SerIle Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr TyrGlu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser LysAsn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg HisArg Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro GluAsn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr ProMet Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met LeuLeu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser AspLeu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser ProGly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His PheArg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro GluSer Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr AlaAla Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880 ThrSer Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser SerTrp 945 950 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu PheLys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp AsnAla Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys ThrSer Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp GlyPro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln AsnIle Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr Pro LeuIle His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala Thr Ala LeuArg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr Ser Ser Lys AsnMet Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080 Pro Ile Pro ProAsp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095 Met Leu PheLeu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110 Gly LysAsn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125 LeuVal Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 11501155 Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 11601165 1170 Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His1175 1180 1185 Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys GluThr 1190 1195 1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His ThrVal Thr 1205 1210 1215 Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu LeuSer Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala TyrAla Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser ThrAsn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys Lys GlyGlu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln Thr Lys GlnIle Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg Ile Ser Pro AsnThr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln Arg Ser Lys Arg AlaLeu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320 Glu Thr Glu Leu GluLys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335 Gln Trp Ser LysAsn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 Gln Ile AspTyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365 Pro LeuSer Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380 AsnArg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 14051410 Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 14151420 1425 Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu1430 1435 1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln ArgGlu 1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val ThrTyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp LeuPro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val HisIle Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr Ser Asn GlySer Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly Ser Leu Leu GlnGly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn Glu Ala Asn Arg ProGly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val Ala Thr Glu Ser Ser AlaLys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560 Pro Leu Ala Trp Asp AsnHis Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575 Glu Trp Lys Ser GlnGlu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 Lys Lys Asp ThrIle Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605 Ala Ile AlaAla Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val ThrTrp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 AsnPro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 16601665 Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 16701675 1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met SerSer 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly SerVal Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr AspGly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn GluHis Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile Arg Ala Glu Val GluAsp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg Asn Gln Ala Ser Arg ProTyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu Ile Ser Tyr Glu Glu Asp GlnArg Gln Gly Ala Glu Pro Arg 1790 1795 1800 Lys Asn Phe Val Lys Pro AsnGlu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815 Val Gln His His Met AlaPro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830 Ala Trp Ala Tyr PheSer Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845 Ser Gly Leu IleGly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860 Asn Pro AlaHis Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875 Phe PheThr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890 AsnMet Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 19151920 Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 19251930 1935 Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile1940 1945 1950 His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg LysLys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro GlyVal Phe 1970 1975 1980 Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly IleTrp Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly Glu His Leu His Ala GlyMet Ser Thr Leu 2000 2005 2010 Phe Leu Val Tyr Ser Asn Lys Cys Gln ThrPro Leu Gly Met Ala 2015 2020 2025 Ser Gly His Ile Arg Asp Phe Gln IleThr Ala Ser Gly Gln Tyr 2030 2035 2040 Gly Gln Trp Ala Pro Lys Leu AlaArg Leu His Tyr Ser Gly Ser 2045 2050 2055 Ile Asn Ala Trp Ser Thr LysGlu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070 Asp Leu Leu Ala Pro MetIle Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085 Ala Arg Gln Lys PheSer Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100 Met Tyr Ser LeuAsp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115 Ser Thr GlyThr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130 Gly IleLys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145 IleArg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 21702175 Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 21802185 2190 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro GlnVal 2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln LysThr Met 2225 2230 2235 Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys SerLeu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys Glu Phe Leu Ile Ser SerSer Gln Asp Gly 2255 2260 2265 His Gln Trp Thr Leu Phe Phe Gln Asn GlyLys Val Lys Val Phe 2270 2275 2280 Gln Gly Asn Gln Asp Ser Phe Thr ProVal Val Asn Ser Leu Asp 2285 2290 2295 Pro Pro Leu Leu Thr Arg Tyr LeuArg Ile His Pro Gln Ser Trp 2300 2305 2310 Val His Gln Ile Ala Leu ArgMet Glu Val Leu Gly Cys Glu Ala 2315 2320 2325 Gln Asp Leu Tyr 2330

1-10. (Canceled).
 11. Modified human factor VIII cDNA, wherein at leastone replacement of a first codon of wild-type human factor VIII cDNA ismade, said first codon encoding a differently charged amino acid thanthe corresponding amino acid of the porcine factor VIII, and whereinsaid at least one replacement is chosen from: (a) a first codon encodinga neutral amino acid is replaced with a second codon that encodes anamino acid with the same charge as the corresponding porcine factor VIIIamino acid; and (b) a first codon encoding a charged amino acid isreplaced with a second codon that encodes an amino acid that is neutralor the same charge as the corresponding porcine factor VIII amino acid.12. The modified human factor VIII cDNA of claim 11, wherein theB-domain is partially or completely deleted.
 13. The modified humanfactor VIII cDNA of claim 12, wherein the deleted B-domain, or segmentthereof, is replaced by a DNA linker segment.
 14. The modified humanfactor VII cDNA of claim 11, further comprising at least onetranscriptional regulatory element.
 15. The modified human factor VIIcDNA of claim 14, wherein the at least one transcriptional regulatoryelement is a dominant selectable marker.
 16. A composition comprisingthe modified human factor VIII cDNA of claim 11 and a pharmaceuticallyacceptable carrier.
 17. A recombinant vector comprising the modifiedhuman factor VIII cDNA of claim
 11. 18. A recombinant host cellcomprising the recombinant vector of claim
 17. 19. A recombinant hostcell comprising the modified human factor VIII cDNA of claim
 11. 20.Modified human factor VIII cDNA, wherein at least one replacement of afirst codon of a wild-type human factor VIII cDNA is made, said firstcodon encoding a differently charged amino acid than the correspondingamino acid of a mutant human factor VIII, and wherein said at least onereplacement is: (a) a first codon encoding a charged amino acid isreplaced with a second codon that encodes an amino acid of the oppositecharge as the corresponding mutant human factor VIII amino acid.
 21. Themodified human factor VIII cDNA of claim 20, wherein the B-domain ispartially or completely deleted.
 22. The modified human factor VIII cDNAof claim 21, wherein the deleted B-domain, or segment thereof, isreplaced by a DNA linker segment.
 23. The modified human factor VIIIcDNA of claim 20, further comprising at least one transcriptionalregulatory element.
 24. The modified human factor VIII cDNA of claim 23,wherein the at least one transcriptional regulatory element is adominant selectable marker.
 25. A composition comprising the modifiedmutant human factor VIII cDNA of claim 20 and a pharmaceuticallyacceptable carrier.
 26. A recombinant vector comprising the modifiedmutant human factor VIII cDNA of claim
 20. 27. A recombinant host cellcomprising the recombinant vector of claim
 26. 28. A recombinant hostcell comprising the modified mutant human factor VIII cDNA of claim 20.29. A method of producing a modified human factor VIII protein,comprising: culturing the host cell of claim 19 or 28 in cell suspensionor on a solid support, as a bath cell culture or as a perfusion cellculture with continuous production of a conditioned medium; andpurifying said protein by chromatographic methods.
 30. The modifiedfactor VIII protein produced by the method of claim
 29. 31. A method oftreating hemophilia A, comprising administering the modified humanfactor VIII cDNA of claim 11 or 20 to at least one patient, andincreasing or maintaining the plasma half-life of the activated,modified human factor VIII protein compared to wild-type human factorVIII protein.
 32. The method of treating hemophilia A as claimed inclaim 31, wherein the plasma half-life of the activated, modified humanfactor VIII protein is more than 3 minutes.
 33. A method of treatinghemophilia A, comprising administering the modified human factor VIIIprotein of claim 30 to at least one patient, and increasing ormaintaining the plasma half-life of the activated, modified human factorVIII protein compared to wild-type human factor VIII protein.
 34. Themethod of treating hemophilia A as claimed in claim 33, wherein theplasma half-life of the activated, modified human factor VIII protein ismore than 3 minutes.
 35. A modified human factor VIII protein comprisingat least one mutation selected from the group consisting of A284K,D318G, M337R, N340D, D349N, N364D, D403S, E434V, E440K, Q468K, R484S,R489G, R583Q, A599 D, E604Q, and G1948K.